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1 한작지 (Korean J. Crop Sci.), 63(1): 35~40(2018) DOI : ISSN (Print) ISSN (Online) Original Research Article 국내산쌀보리맥강을이용한 glutamate 로부터 GABA 생산 이미자 1, 김신혜 1 김형순 2 김현영 1 서우덕 1 최식원 1 이광식 1 장기창 1 Production of γ-aminobutyric Acid Using the Korean Hull-less Barley Bran with Mi Ja Lee 1,, Shin Hye Kim 1, Hyung Soon Kim 2, Hyun Young Kim 1, Woo Duck Seo 1, Sik Won Choi 1, Kwang Sik Lee 1, and Ki Chang Jang 1 ABSTRACT γ-aminobutyric acid (GABA) has antihypertensive and anti-stress effects on humans. The present study aimed to investigate the effect of glutamate concentration, bran content, and barley cultivars on GABA production and determine the optimal reaction condition. Barley bran resulted in higher GABA productivity compared with that of rice bran. The higher the bran content, the higher was the GABA productivity. Furthermore, high glutamate concentration resulted in high GABA production. However, there was a decrease in the glutamate conversion rate. The production of GABA varied with temperature and barley cultivar. The optimal condition for GABA production using barley bran was 0.15 g/ml barley content, 10 mm glutamate concentration, and 20 C reaction temperature. Under optimal condition, the GABA concentration was mm, and glutamate conversion rate was 75.1%. Furthermore, the GABA productivity of the GABA production reaction using barley bran differed depending on the cultivar. Dasong and Yeongbaekchal showed higher GABA productivity than that by other cultivars. Keywords : γ-aminobutyric acid, barley bran, rice bran γ-aminobutyric acid (GABA) 는 4개의탄소를중심으로구성되어있는비단백질구성아미노산으로뇌와척추에존재하며신경전달물질로서혈류개선을통한뇌의산소공급을원활하게하여뇌의대사를촉진시키고, 결합하는수용체의종류에따라신경억제를통한불안감해소, 기억력증강또는조울증등에영향을준다 (Shin et al., 2007; Lim et al., 2009). 또한스트레스해소, 혈압강하작용, 우울증개선, 중풍과치매예방, 불면, 비만, 갱년기장애등에효과가있는것으로알려져있으며, 최근의연구에서는 Alzheimer s disease (Rissma et al., 2007), 불안장애 (Obata et al., 2008), 우울증 (Hasler et al., 2007) 등의질병이 GABA성신경세포 (GABAergic neurons) 의 GABA결핍에원인이있는것으로보고되고있다. 최근의연구는이러한질병의치료에사용하기위한기능성식품에관심이집중되고있고, GABA 는다양한식품속에포함되어있으나함량이낮아자연적인섭취로 GABA의생리작용을기대하기는어려워 GABA 의대량생산을위해인위적으로함량을높이는연구가진행되고있다. 녹차 (Jeng et al., 2007), 현미 (Komatsuzaki et al., 2007), 보리 (Chung et al., 2009), 밀 (Young et al., 2011), 홍국미 (Chuang et al., 2011) 등다양한종류의식물을활용한유산균발효를통하여 GABA생산성을향상시키는연구들이수행되었으며, monosodium glutamate (MSG) 를첨가하여 GABA 생성량을증가시키는효과에대한연구또한활발히이뤄지고있다 (Choi et al., 2006) 맥강과미강은보리와쌀의도정과정중발생하는부산물로연간생산량은맥강은 6만2천톤, 미강은 10만톤에이르고있다 (Kim et al., 2004). 이러한부산물들은많은섬유소와생리활성물질들을포함하고있지만일부가사료또 1) 농촌진흥청국립식량과학원작물기초기반과 (Crop Foundation Division, National Institute of Crop Science, 181 Hyeoksin-ro, Iseo-myeon, Wanju-Gun, Jeollabuk-do , Korea) 2) 서남대학교환경화학공학과 (Department of Environmental & Chemical Engineering, Seonam University, Namwon , Korea) Corresponding author: Mi Ja Lee; (Phone) ; ( ) esilvia@korea.kr <Received 21 October, 2017; Revised 4 January, 2018; Accepted 18 January, 2018> c 본학회지의저작권은한국작물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 36 한작지 (KOREAN J. CROP SCI.), 63(1), 2018 는퇴비로활용되고있을뿐, 대부분폐기되고있는실정이다. 맥강과미강으로부터 polyphenol 및 xylose등과같은여러가지기능성물질들의추출에대한연구들이이루어졌지만 GABA생성을위한곡물부산물의활용은거의연구되어있지않다. 본연구에서는곡류가공부산물인맥강과미강에포함되어있는 GAD를활용하여 glutamate를 GABA로전환하는반응에서 GABA의생산성향상을위한최적반응조건을알아보고자맥강과미강의첨가량, glutamate 농도, 반응온도및국내산쌀보리품종에따른 GABA의생산성을비교하였다. 재료및방법시험재료본실험에사용한맥강은농촌진흥청에서육성한쌀보리 13품종을국립식량과학원시험포장에서표준재배법에준하여재배하였으며, 2015년중순에파종하고, 2016년 6월에수확하여, 14 저장고에저장하였다. 이들품종들의특성은 Table 1에나타내었으며, 이를사다께도정기 (Sadake test mill, M05, Tokyo, Japan) 를이용하여 73% 도정하여사용하였다. 미강은 ( 주 ) 산마을 ( 경남창녕군장마면 ) 에서구입하였다. GABA와 monosodium glutamate 는 Sigma Chemicals Co. (St. Louis, MO, USA) 에서구입하였으며, 그외모든시약은 1급이상시약을사용하였다. 의 GABA 전환반응 의 GABA전환실험은 10 mm glutamate 용액에맥강 0.15 g/ml를첨가하여 20 에서 6시간동안반응시켰으며, 반응후 80 에서 20분동안처리하여효소활성을정지시키고, 원심분리하여상등액을분석에사용하였다. GABA 및 glutamic acid 함량분석 GABA 및 glutamic acid 함량은 AccQ Tag UPLC (Waters, Milford, MA) 분석시스템을이용하여 Limure et al. (2009) 의방법에기초하여측정하였다. 시료는멤브레인필터를이용하여여과하였으며, 여과한시료는 UPLC분석을하기위하여 AccQ-Tag 방법을이용하여유도체화하였다. UPLC분석시스템은 Waters Acquity를사용하였고, 컬럼은 AccQ Tag ultra amino acid analysis column ( mm; Waters), 컬럼온도는 49, 시료온도는 20 였으며, 검출기는 FLR Detector (Waters) 로파장은 EX. 266 nm, EM. 473 nm이었다. 이동상은 A는 100% AccQ Tag ultra UPLC amino acid analysis eluent A, B는 10% AccQ Tag ultra UPLC amino acid analysis eluent B, C는 100% 증류수, D는 100% AccQ Tag ultra UPLC amino acid analysis eluent B를기울기용리하였으며, 유속은 0.7 ml/min이었고, 주입량은 1 μl이었고, 각시료는 3반복으로측정하였다. 크로마토그램데이터는 Empower personal software (Waters) 를사용하여분석하였으며, GABA 를제외한아미노산함량분석은 Waters amino acids standards (Waters) 를이용하여분석하였다. Table 1. Characteristics of different hull-less barley cultivars. Saessal Jinmichapssal Cheonghossal Duwonchapssal Jaeanchalssal Dasongssal Chalssal Boseokchal Heuknarae Yeongbaekchal Betaone Saechal Daeanchal Saessal Cultivar Characteristics Normal starch, 6-rowed Low amylose starch (waxy), 2-rowed Normal starch, 6-rowed Normal starch, 2-rowed, purple lema Normal starch, 6-rowed, black lema Low amylose starch (waxy), 2-rowed Normal starch, 6-rowed

3 국내산쌀보리맥강을이용한 glutamate 로부터 GABA 생산 37 통계처리실험치는평균값과표준오차로표시하였다. 통계분석은 SAS Enterprise Guide 4.0 (Statistical analysis system, 2016, Cray, NC, USA) 로분석하고, 시료간의유의적인차이는 Duncan s multiple range test로유의수준 5% (p<0.05) 에서검증하였다. 결과및고찰미강및맥강첨가량변화에따른 GABA 생산미강및새찰쌀보리맥강을활용한 GABA생산반응에서이들첨가량에의한 GABA생산효과를알아보리위하여 10 mm glutamate용액 10 ml에미강과새찰쌀보리맥강을각각 0.025, 0.05, 0.1, 0.15 및 0.2 g/ml를첨가하고 20 에서 6시간반응시킨후 GABA와 glutamic acid 함량변화를확인하였다. Fig. 1에서보는바와같이미강과맥강첨가량이증가할수록 GABA 생산량은유의적으로증가하였다. 미강의첨가량에따른 GABA 생산량은 mm 범위였으며, 첨가량이 0.2 g/ml까지 GABA 생산량은지속적으로증가하였다. 맥강의경우에는 mm 범위로맥강첨가량이증가함에따라 GABA 생산량이급속하게증가하여맥강첨가량이 0.15 g/ml에서최대 GABA 생산량을나타내었고이후에소량감소하였다. 미강과맥강첨가량이 0.2 g/ml 이상의경우에는교반이어려워져실험을할수없었다. Glutamic acid 함량은 GABA 생산량과는반대로미강과맥강첨가량이증가함에따라감소하는경향을보였다. 맥강에의한 GABA 함량이최대인 0.15 g/ml 에서전환되지않고남아있는 glutamic acid의함량은 2.78 mm이었으며, 미강의경우에는미강첨가량이 0.2 g/ml에 서 glutamic acid는 mm이전환되지않고남아있었다. 실험결과맥강이미강보다약 2-3배높은 GABA 생산량을보였고, GABA 최대생산을위한맥강의최적첨가량은 0.15 g/ml 이었으며, 이러한결과는 Limure et al. (2009) 이보고한결과와일치하였다. 또한, Limure et al. (2009) 은만약반응기의용량이증가된다면맥강첨가량의증가가가능할것이고이에따라 GABA 생산량도높아질수있을것이라고보고하였는데본실험에서도반응기의용량이증가된다면맥강첨가량이 0.2 g/ml 이상에서도교반이가능할것으로생각된다. 농도에따른 GABA 생산일반적으로 GABA를합성하는공정은 GAD 효소의촉매작용에의해 glutamate를탈탄산화시켜 GABA로만드는과정이다. GABA 생산량을향상시키기위해서초기 glutamate 농도를 10, 20, 30, 40 mm로변화시켜반응시켰으며, 이때미강과맥강첨가량은 0.15 g/ml이었고, 20 에서 6시간반응시켰다. 실험결과생성된 GABA 함량과반응하지않고남아있는 glutamic acid 함량및 glutamate 전환율을 Table 2에나타내었다. 시험재료인맥강과미강에포함되어있는 GABA 함량은각각 1.22와 1.28 mm이었으며, glutamic acid 함량은각각 0.48과 1.27 mm이었다. 농도변화에따른 GABA 생산량은맥강과미강모두 glutamate 농도가 20 mm 까지는 GABA 생산량이증가하였으나그이상농도에서는유의적인증가는이루어지지않았다. 맥강의경우 GABA 생산량은 20 mm에서높았지만 glutamate 전환율은 10 mm에서가장높았으며, 미강의경우에도 glutamate 농도가 10 mm에서전환율이가장높았으며그이상의농 (A) (B) Fig. 1. Effect of the initial bran content on the production of γ-aminobutyric acid (GABA) from glutamate using Saechal barley and rice bran. (A) GABA content, (B) Glutamic acid content. The solution containing barley and rice bran, and 10 ml of distilled water with 10 mm glutamate was incubated at 20 C for 6 h.

38 한작지 (KOREAN J. CROP SCI.), 63(1), 2018 Table 2. Effect of the initial glutamate concentration on the production of γ-aminobutyric acid (GABA) from glutamate using Saechal barley and rice brans.

48 b 7.21±1.83 d 27.9 20 24.58±2.29 a 11.28±1.05 b 43.6 2.60±0.34 a 21.46±1.99 c 0.0 30 20.99±8.66 a 14.76±5.82 b 50.8 2.61±0.58 a 28.71±4.95 b 4.3 40 25.87±3.56 a 26.68±3.07 a 33.3 2.66±0.32 a 39.

4 38 한작지 (KOREAN J. CROP SCI.), 63(1), 2018 Table 2. Effect of the initial glutamate concentration on the production of γ-aminobutyric acid (GABA) from glutamate using Saechal barley and rice brans. cont. GABA Barley bran Glutamic acid conversion rate (%) GABA Rice bran Glutamic acid conversion rate (%) ±0.11 c1) 0.48±0.06 c 1.28±0.13 b 1.27±0.04 e ±0.77 b 2.49±0.19 c ±0.48 b 7.21±1.83 d ±2.29 a 11.28±1.05 b ±0.34 a 21.46±1.99 c ±8.66 a 14.76±5.82 b ±0.58 a 28.71±4.95 b ±3.56 a 26.68±3.07 a ±0.32 a 39.88±4.81 a 0.3 1) Mean value of triplicates ± standard deviation. Values with different letters in the same row differ significantly from each other (p<0.05). 도에서는대부분이전환하지않고그대로남아있었으며맥강이미강보다훨씬높은 GABA 생산효율을보였다. 그러므로본실험의경우 GABA 생산을위한최적 glutamate 농도는 10 mm 이었다. Liu et al. (2005) 과 Saikusa et al. (1994) 은쌀의침지중 GABA 축적은 GAD활성과관계가있으며, 발아보리에서의 GABA 함량이발아된밀이나쌀보다도높다고보고하였다. 본실험에서는 GAD활성을조사하지는않았지만실험결과맥강이미강보다훨씬높은 GABA 생산을보여 Ito (2004) 와 Kihara et al. (2007) 의보고와같이미강보다맥강의 GAD 활성이높을것으로생각된다. GABA 생산에서품종과온도의영향미강보다높은 GABA 생산량을나타낸맥강을활용하기위하여다른국내산보리품종의맥강이 GABA 생산에미치는영향을알아보았다. 국내주요쌀보리품종중메성쌀보리인새쌀과찰성쌀보리인새찰쌀및단백질과라이신 함량이높은찰성쌀보리인대안찰쌀보리의맥강을이용하여 GABA 생산실험을하였다 (Fig. 2). 이들맥강은 200 g을 test mill (TM-05C, SATAKE, Japan) 로 154 g까지도정하고남은부산물이다. GABA 생산량은 20 에서 6시간반응시킨것 ( mm) 이 40 에서반응시킨것 ( mm) 보다모든품종에서높았으며, 20 에서 6시간반응의경우품종별로유의적인차이를나타내었고, 새쌀보리 (6.86 mm) 가새찰쌀보리 (4.76 mm) 와대안찰쌀보리 (2.13 mm) 보다높은 GABA 생산량을보였다. 전환되지않고남은 glutamic acid 함량은 20 반응에서는 mm이었으며, 40 반응의경우에는 mm로 40 반응의경우전환되지않고남은 glutamic acid 함량이 20 반응에서보다높아 GABA 생산반응은 40 보다는 20 가적합하였다. Paidaeng et al. (2014) 은 brown rice를발아시키고이때 GABA와 glutamic acid 함량을조사하였는데, rice의품종에따라 GAD활성과 glutamic acid 함량이다르며 GABA 생산량은이들과깊은관계가있다고보고하였다. 본연구 (A) (B) Fig. 2. Effect of barley cultivar on the production of γ-aminobutyric acid (GABA) from glutamate using barley bran. (A) GABA content, (B) Glutamic acid content. The solution containing 0.15 g/ml barley bran and 10 ml distilled water with 10 mm glutamate was incubated.

국내산쌀보리맥강을이용한 glutamate 로부터 GABA 생산 39 에서는보리품종들이 GABA 생산반응에미치는영향을알아보기위하여국내산주요쌀보리 13품종을도정하여얻어진맥강 0.15 g/ml, 10 mm glutamate 용액에서 20, 6시간반응시키고생성된 GABA 농도와반응하지않고남은 glutamic acid 농도를분석하였다.

87 mm로시험품종중에서비교적가장높은 GABA 생산성을나타내었으며, 진미찹쌀은 0.79 mm로가장낮은 GABA 생산성을나타내었다. 반응하지않고남은 glutamic acid 함량은 Fig. 4에나타내었다. Glutamic acid 함량은 1.33-6.09 범위였으며, GABA 생산성이높았던다송 (1.69 mm) 과영백찰쌀보리 (2.

5 국내산쌀보리맥강을이용한 glutamate 로부터 GABA 생산 39 에서는보리품종들이 GABA 생산반응에미치는영향을알아보기위하여국내산주요쌀보리 13품종을도정하여얻어진맥강 0.15 g/ml, 10 mm glutamate 용액에서 20, 6시간반응시키고생성된 GABA 농도와반응하지않고남은 glutamic acid 농도를분석하였다. 그결과 GABA 함량은보리품종에따라 범위로품종별로큰차이를나타내었다 (Fig. 3). 찰성쌀보리품종들의평균 GABA 생산량은 4.68 mm 이었으며, 메성쌀보리품종들의평균 GABA 생산량은 4.90 mm이었으나찰성과메성품종들간의뚜렷한 경향은없었다. 다송은 7.40 mm과영백찰은 7.87 mm로시험품종중에서비교적가장높은 GABA 생산성을나타내었으며, 진미찹쌀은 0.79 mm로가장낮은 GABA 생산성을나타내었다. 반응하지않고남은 glutamic acid 함량은 Fig. 4에나타내었다. Glutamic acid 함량은 범위였으며, GABA 생산성이높았던다송 (1.69 mm) 과영백찰쌀보리 (2.44 mm) 가비교적낮은 glutamic acid함량을나타내었다. 그러나통계처리결과 GABA 생산량은 glutamic acid 함량과유의적인상관관계를나타내지는않았다 (r=-0.324, p<0.1). Fig. 3. Production of γ-aminobutyric acid (GABA) from glutamate by barley bran which was produced from various cultivars. The solution containing 150 mg/ml barley bran and 10 ml distilled water with 10 mm glutamate was incubated for 6 h at 20 C. Fig. 4. Concentration of glutamic acid in the production reaction of γ-aminobutyric acid (GABA) from glutamate by barley bran which was produced from various cultivars. The solution containing 150 mg/ml barley bran and 10 ml distilled water with 10 mm glutamate was incubated for 6 h at 20 C.

6 40 한작지 (KOREAN J. CROP SCI.), 63(1), 2018 적요 곡류가공부산물인미강과맥강을활용한 GABA 생산성향상을위하여맥강과미강의첨가량, glutamate 농도및보리품종에따른 GABA 생산량을측정비교하였다. 그결과맥강이미강보다모든조건에서높은 GABA 생산성을보였고, 맥강을활용한 GABA 생산반응에서최적조건은맥강첨가량은 0.15 g/ml, glutamate 농도는 10 mm, 반응온도는 20 로이때 glutamate의 GABA로의전환율은 75.1% 였다. 또한보리품종에따른 GABA 생산성은유의적인차이를나타내었으며다송과영백찰쌀보리가높은 GABA 생산성을나타내었다. 본연구결과부산물인맥강을이용하여 GABA 생산성을향상시킬수있었으며, 보리품종이 GABA 생산의중요요인으로작용하였다. 사사 본논문은농촌진흥청연구사업 ( 세부과제명 : 곡류가공부산물이용 GABA 생산기술개발, 세부과제번호 :PJ ) 의지원에의해수행되었습니다. 인용문헌 (REFERENCES) Choi, S. I., J. W. Lee, S. M. Park, M. Y. Lee, G. E. Ji, M. S. Park, and T. R. Heo Improvement of γ-aminobutyric acid (GABA) production using cell entrapment of Lactobacillus brevis GABA 057. J. Microbiol. Biotechnol. 16 : Chuang, C. Y., Y. C. Shi, H. P. You, Y. H. Lo, and T. M. Pan Antidepressant effect of GABA-rich monascus-fermented production forced swimming rat model. J. Agric. Food Chem. 59 : Chung, H. J., S. H. Jang, H. Y. Cho, and S. T. Lim Effects of steeping and anaerobic treatment on GABA (γ-aminobutyric acid) content in germinated waxy hull-less barley. LWT-Food Sci. Technol. 42 : Hasler, G., J. Van der Veen, T. Tumonis, N. Meyers, J. Shen, and W. C. Drevets Reduced prefrontal glutamate/glutamine and γ-aminobutyric acid levels in major depression determined using proton magnetic resonance spectroscopy. Archiv. General Psychiatry. 64 : Ito, S Marketing of value added rice products in Japan: Germinated brown rice and rice bread. pp In; FAO rice conference. February 12, Rome, Italy. Jeng, K. C., C. S. Chen, Y. P. Fang, R. C. W. Hou, and Y. S. Chen Effect of microbial fermentation on content of statin, GABA and polyphenols in Pu-Erh tea. J. Agric. Food Chem. 55 : Kihara, M., Y. Okada, T. Limure, and K. Ito Accumulation and degradation of two functional constituents, GABA and β-glucan and their varietal differences in germinated barley grains. Breeding Sci. 57 : Kim, Y. H., C. S. Kang, and Y. S. Lee Quantification of tocopherol and tocotrienol content in rice bran by near infrated reflectance spectroscopy. Korean J. Crop Sci. 43 : Komatsuzaki, N., K. Tsukahara, H. Toyoshima, T. Suzuki, N. Shimizu, and T. Kimura Effect of soaking and gaseous treatment on GABA content in germinated brown rice. J. Food Eng. 78 : Lim, S. D., S. H. Yoo, H. D. Yang, S. K. Kim, and S. Y. Park GABA productivity in yogurt fermented by freeze dried culture preparations of Lactobacillus acidophilus RMK567. Korean J. Food Sci. An. 29 : Limure, T., M. Kihara, N. Hirota, T. Zhou, K. Hayashi, and K. Ito A method for production of γ-amino butyric acid (GABA) using barley bran supplemented with glutamate. Food Research International. 42 : Liu, L. L., H. Q. Zhai, and J. M. Wan Accumulation of γ-aminobutyric acid in giant-embryo rice grain in relation to glutamate decarboxylase activity and its gene expression during water soaking. Cereal Chemistry. 82 : Obata, K., M. Hirono, N. Kume, Y. Kawaguchi, S. Itohara, and Y. Yanagawa GABA and synaptic inhibition of mouse cerebellum lacking glutamate decarbosylase67. Biochem. Biophy. Res. Commun. 370 : Paidaeng, K., C. Ninnart, P. Rath, and C. Saiwarun Gamma-aminobutyric acid and glutamic acid contents, and the GAD activity in germinated brown rice (oryza sativa L.): effect of rice cultivars. Food Sci. Biotechnol. 23 : Rissma, R. A., A. L. De Blas, and D. M. Amstrong GABA receptors in aging and Alzheimer s disease. J. Neurochem. 103 : Saikusa, T., T. Horino, and Y. Mori Accumulation of γ-aminobutyric acid (Gaba) in the rice germ during water soaking. Biosci. Biotechnol. and Biochem. 58 : Shin, J. W., D. G. Kim, Y. W. Lee, H. S. Lee, K. S. Shin, C. S. Choi, and G. S. Kwon Isolation and characterization of Lactobacillus brevis AML15 producing γ-aminobutyric acid. J. Life Sci. 17 : Takashi, L., M. Kihara, N. Hirota, T. Zhou, K. Hayashi, K Ito A method for production of γ-amino butyric acid (GABA) using barley bran supplemented with glutamate. Food Resear. Int. 42 : Young, Y. S., J. K. Park, H. D. Hang, and Y. W. Rhee Sequential hydration with anaerobic and heat treatment increases GABA (γ-aminobutyric acid) content in wheat. Food Chem. 129 :

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THE JOURNAL OF KOREAN INSTITUTE OF ELECTROMAGNETIC ENGINEERING AND SCIENCE. 2018 Oct.; 29(10), 799 804. http://dx.doi.org/10.5515/kjkiees.2018.29.10.799 ISSN 1226-3133 (Print) ISSN 2288-226X (Online) Method